A method for culturing Newcastle disease virus using BHK-21 fully suspended cells.

Abstract

This application provides a method for culturing Newcastle disease virus (NDV) in BHK-21 fully suspended cells, belonging to the field of vaccine preparation technology. This method employs a two-stage pH control approach to improve NDV culture titer and immunogenicity. The two-stage pH control involves maintaining the pH at 7.2-7.4 during the BHK-21 cell growth stage and at 6.2-6.8 during the NDV amplification stage. The NDV titer and immunogenicity cultured using this method achieve levels comparable to those obtained using chicken embryo culture.

Description

Technical Field

[0001] This invention belongs to the field of vaccine preparation technology, specifically relating to a method for culturing Newcastle disease virus using BHK-21 fully suspended cells. Background Technology

[0002] Newcastle disease (ND) in chickens is a highly contagious disease caused by Newcastle disease virus (NDV). It has an extremely high mortality rate and causes enormous economic losses to the poultry industry worldwide. The World Organisation for Animal Health (OIE) classifies it as a Category A disease, while my country classifies it as a Category I animal disease. Currently, vaccination remains the most effective and economical means of preventing ND outbreaks.

[0003] In my country, Newcastle disease (ND) vaccine production primarily utilizes the traditional chicken embryo process. While this process still plays a crucial role in ND vaccine control, embryo virus production is labor-intensive and suffers from drawbacks such as long production cycles, susceptibility to contamination, and the presence of numerous extraneous proteins. Especially during NDV pandemics, the surge in vaccine demand and the impact of the epidemic led to a severe shortage of SPF chicken embryos, limiting production capacity. Therefore, a new NDV production process based on animal cell suspension culture technology has emerged.

[0004] Cultured viruses in fully suspended mammalian cells is the mainstream method for producing whole-virus inactivated vaccines both domestically and internationally, and has been applied in poultry vaccine production in recent years. Using fully suspended cells to replace chicken embryos in the production of Newcastle disease (ND) vaccines overcomes the shortcomings of chicken embryo production processes, offering advantages such as controllable culture conditions, good product uniformity, high vaccine quality, and the ability to achieve large-scale production. It also reduces overall costs related to energy consumption, space, equipment, and labor. Cell lines used for animal vaccine production mainly include Vero cells, MDCK cells, ST cells, and DF-1 cells. In recent years, research on NDV culture processes both domestically and internationally has focused on Vero cells and BHK-21 cells. Studies have found that BHK-21 cells are more suitable for the growth of NDV type VII compared to Vero cells, and BHK-21 cells can be trained to grow in suspension, and have been successfully used in the production of veterinary vaccines and human recombinant proteins. However, compared with chicken embryos as a virus culture medium, the use of BHK-21 fully suspended cells to culture Newcastle disease virus in large-scale bioreactors still has the problem that the virus titer and immunogenicity are lower than those of embryo virus. Therefore, it is essential to conduct research on large-scale bioreactor processes.

[0005] Current optimization of NDV cell culture processes focuses on temperature, trypsin concentration, serum concentration, infection time, and multiplication factor, with limited optimization of pH. Studies on NDV culture in suspension cells have not included pH optimization. Only Dong Bingmei et al. optimized pH in their BHK-21 adherent NDV culture process, with an optimization range of 7.0-8.0, showing an optimal pH of 7.2 (non-patent literature, Optimization of Proliferation Conditions of Newcastle Disease Virus La Sota Strain in BHK-21 Cells, *Chinese Journal of Animal Husbandry and Veterinary Medicine*, 2019, Vol. 46, No. 3). This suggests that culture pH generally has little impact on NDV viral titer and immunogenicity, and that the optimal pH is above 7.0. Studies have found that Newcastle disease virus (NDV) requires low pH to enter certain cells. Short-term treatment with a low-pH buffer (pH 5.0) increased NDV fusion with cells by 30%, thereby promoting viral entry into cells (non-patent literature, Lorena S et al., 2014, Entry of Newcastle Disease Virus into the host cell: Role of acidic pH and endocytosis. Biochim Biophys Acta Biomembr. 1838:300-309). Therefore, the limited research on pH optimization in suspension cell culture processes may be due to consideration of only the pH range required to maintain the physiological activity of cells and viruses, without taking into account the pH required for viral infection, replication, and release within cells.

[0006] In view of the above, this application is hereby submitted. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this application provides a method for culturing Newcastle disease virus (NDV) in BHK-21 fully suspended cells. This method uses two-stage pH control to improve NDV culture titer and immunogenicity, achieving virus titer and immunogenicity levels comparable to those of chicken embryo culture.

[0008] This application provides a method for culturing Newcastle disease virus in BHK-21 fully suspended cells. The method employs two-stage pH control, wherein the pH is controlled at 7.2-7.4 during the BHK-21 cell growth stage and at 6.2-6.8 during the Newcastle disease virus amplification stage. Preferably, the pH is controlled at 6.4-6.6.

[0009] Preferably, the two-stage pH control is adjusted by introducing CO2 or adding sodium bicarbonate during the culture process.

[0010] The method for culturing Newcastle disease virus using BHK-21 fully suspended cells is characterized by comprising the following steps:

[0011] Step (1) Establishment of Newcastle disease virus seed batch;

[0012] Step (2) BHK-21 fully suspended cell culture;

[0013] Step (3) NDV virus amplification and harvesting.

[0014] The establishment of the Newcastle disease virus seed batch in step (1) includes inoculating BHK-21 fully suspended cells with Newcastle disease virus at an MOI of 0.01-0.1, adding trypsin at a final concentration of 20-50 mg / L, culturing in a 37°C, 5% CO2 shaking incubator for 72 h, harvesting the virus solution, and passaged 3-4 times to use as the reactor seed virus.

[0015] Step (2) of BHK-21 full suspension cell culture includes thawing frozen cells from a liquid nitrogen tank, gradually scaling up to obtain a sufficient quantity of BHK-21 cells, and maintaining a cell density of 1.0 × 10⁻⁶ cells. 6 -1.5×10 6 Cells were inoculated at a rate of 1 / ml into the bioreactor. The bioreactor temperature was set at 37℃. The dissolved oxygen (DO) value was controlled at 30%-50% by stirring speed and aeration rate. The pH was controlled at 7.2-7.4 by introducing CO2 or adding sodium bicarbonate. The cells were cultured for 72-96 hours, and the cell density and viability were monitored.

[0016] Step (3) NDV virus amplification and harvesting includes when the cell density is ≥1.0×10⁻⁶. 7 When the cell density was adjusted to 5.0 × 10⁶ cells / ml, BHK203V virus amplification medium was added. 6 -6.0×10 6 Inoculate with chicken Newcastle disease virus seed at a concentration of 1 virus / ml, with an MOI of 0.01-0.1. Simultaneously add trypsin to a final concentration of 20-50 mg / L. Set the reactor temperature to 32-35℃, dissolved oxygen to 30%-50%, and control the pH at 6.2-6.8 by introducing CO2 or adding sodium bicarbonate, preferably 6.4-6.6. Harvest the virus solution after 96-120 hours of incubation.

[0017] Steps (2) and (3) are carried out in a 14L bioreactor.

[0018] Optionally, the Newcastle disease virus in chickens is the N7a strain.

[0019] This application also provides chicken Newcastle disease virus prepared by the above method.

[0020] This application also provides the application of the Newcastle disease virus obtained by the above technical solution in the preparation of Newcastle disease inactivated vaccine, Newcastle disease trivalent inactivated vaccine, Newcastle disease adenovirus quadrivalent inactivated vaccine, Newcastle disease nephrotic syndrome quadrivalent inactivated vaccine, and Newcastle disease nephrotic syndrome pentavalent inactivated vaccine.

[0021] Beneficial effects of the present invention

[0022] 1. This application demonstrates that the two-stage pH control significantly improved the NDV virus titer and HI titer in BHK-21 fully suspended cell culture, with an HA titer of 10-11 log2 and a viral load of 8.89-9.57 lg TCID. 50 / ml and 8.63-9.38lgEID 50 / 0.1ml, HI titer reaches above 8.0log2, virus titer is not significantly different from chicken embryo process, HI titer is higher than chicken embryo process.

[0023] 2. The Newcastle disease virus prepared in this application can be used in a multivalent vaccine for chickens, producing high levels of HI antibodies, which can completely protect against virulent virus attacks, demonstrating good compatibility between different antigens.

[0024] 3. Compared with vaccines produced from chicken embryos, NDV inactivated vaccines produced by suspension cell culture technology are free from exogenous factor contamination, are easy to scale up, and can better maintain viral stability, thus achieving maximum yield while steadily improving product quality. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0026] This application provides a method for improving the titer and immunogenicity of Newcastle disease virus derived from BHK-21 fully suspended cells. The method employs a two-stage pH control, wherein the pH is controlled at 7.2-7.4 during the BHK-21 cell growth stage and at 6.2-6.8 during the Newcastle disease virus amplification stage. Preferably, the pH is controlled at 6.4-6.6.

[0027] Preferably, the two-stage pH control is adjusted by introducing CO2 or adding sodium bicarbonate during the culture process.

[0028] Virus culture in fully suspended cells requires consideration not only of the pH range needed to maintain the physiological activity of cells and viruses, but also of the pH required for viral infection, replication, and release within cells. This invention corrects the misconception that optimizing virus culture processes in suspended cells only considers maintaining the physiological pH of cells and viruses.

[0029] This invention also provides a method for culturing Newcastle disease virus in BHK-21 fully suspended cells, characterized by comprising the following steps:

[0030] Step (1) Establishment of Newcastle disease virus seed batch;

[0031] Step (2) BHK-21 fully suspended cell culture;

[0032] Step (3) NDV virus amplification and harvesting.

[0033] The method for establishing the Newcastle disease virus seed batch in step (1) is to inoculate BHK-21 fully suspended cells with Newcastle disease virus at an MOI of 0.01-0.1, add trypsin at a final concentration of 20-50 mg / L, and culture in a 37℃, 5% CO2 shaking incubator for 72 h to harvest the virus liquid. The virus is then passaged 3-4 times in the same manner as the reactor seed virus.

[0034] The method in step (2) involves resuscitating frozen cells from a liquid nitrogen tank, gradually scaling up to obtain a sufficient quantity of BHK-21 cells, at a cell density of 1.0 × 10⁻⁶ cells / year. 6 -1.5×10 6 Cells were inoculated at a rate of 1 / ml into the bioreactor. The bioreactor temperature was set at 37℃. The dissolved oxygen (DO) value was controlled at 30%-50% by stirring speed and aeration rate. The pH was controlled at 7.2-7.4 by introducing CO2 or adding sodium bicarbonate. The cells were cultured for 72-96 hours, and the cell density and viability were monitored.

[0035] The method in step (3) is as follows: when the cell density is ≥1.0×10 7 When the cell density was adjusted to 5.0 × 10⁶ cells / ml, BHK203V virus amplification medium was added. 6 -6.0×10 6 Inoculate with chicken Newcastle disease virus seed at a concentration of 1000 / ml and MOI = 0.01-0.1, while adding trypsin at a final concentration of 20-50 mg / L. Set the reactor temperature to 32-35℃ and dissolved oxygen to 30%-50%. Control the pH to 6.2-6.8 by introducing CO2 or adding sodium bicarbonate, preferably 6.4-6.6. Harvest the virus solution after culturing for 96-120 hours.

[0036] Unless otherwise specified, the experimental methods described in this invention are all conventional methods; the biological materials and culture media described herein are all commercially available unless otherwise specified.

[0037] Example 1: Establishment of Newcastle Disease Virus Seed Batch

[0038] The attenuated Newcastle disease virus strain N7a of genotype VII, disclosed in Chinese patent CN107287168A, was inoculated into BHK-21 fully suspended cells at an MOI of 0.01-0.1. Trypsin was added to a final concentration of 20-50 mg / L, and the cells were cultured in a shaking incubator at 37°C and 5% CO2 for 72 hours. The virus solution was then harvested and passaged three times in the same manner as the inoculation reactor.

[0039] Example 2: pH Optimization in BHK-21 Fully Suspension Cell Culture

[0040] Remove the frozen BHK-21 cells from the liquid nitrogen tank and quickly place them in a 37°C water bath, shaking constantly to thaw them rapidly. Add them to a sterile centrifuge tube containing BHK202 fully suspended cell culture medium, centrifuge at 800 rpm for 5 minutes, discard the supernatant, resuspend in BHK202 medium, place in a 125 ml shake flask, add culture medium to a final volume of 15 ml, and incubate for approximately 72 hours. Culturing at a rate of 1.0 × 10⁻⁶ cells / mL... 6 -1.5×10 6 Cells were passaged at a density of [number] cells / ml. Sufficient BHK-21 cells were obtained through 3-5 passages and seeded into a 14L bioreactor at the passage density. The reactor temperature was set at 37℃. During culture, the dissolved oxygen (DO) was maintained at 30%-50% by controlling the stirring speed and aeration rate for 72 hours. Cell density and viability were monitored during the culture. The pH was controlled at 7.6-7.8 (Process 1), 7.4-7.6 (Process 2), 7.2-7.4 (Process 3), 7.0-7.2 (Process 4), 6.8-7.0 (Process 5), 6.6-6.8 (Process 6), and 6.4-6.6 (Process 7) during the BHK-21 cell culture stages. Cell density and viability were measured using a cell counter. The results are shown in Table 1.

[0041] Table 1 Effect of pH on BHK-21 cell density

[0042] The results showed that compared with processes 2, 3, and 4, the cell density and viability were lower in processes 1, 6, and 7, indicating that pH values ​​greater than 7.6 or less than 6.8 had a significant negative impact on the growth of BHK-21 cells. Furthermore, process 3 showed the highest cell density and viability at 72 hours; therefore, the pH should be controlled between 7.2 and 7.4 during the BHK-21 cell culture stage.

[0043] Example 3: pH Optimization during NDV Virus Amplification Phase

[0044] The BHK-21 fully suspended cell culture stage was carried out according to the steps in Example 2, with the pH controlled at 7.2-7.4.

[0045] When cell density ≥1.0×10 7 When the cell density was adjusted to 5.0 × 10⁶ cells / ml, BHK203V virus amplification medium was added. 6 -6.0×10 6 Inoculate with chicken Newcastle disease virus N7a strain at a dose of MOI = 0.01-0.1, and simultaneously add trypsin to a final concentration of 20-50 mg / L. Control the reactor temperature at 32-35℃ and dissolved oxygen at 30%-50%, and incubate for 96-120 hours to harvest the virus solution. During the virus amplification stage, control the pH at 7.0-7.2 (process a), 6.8-7.0 (process b), 6.6-6.8 (process c), 6.4-6.6 (process d), 6.2-6.4 (process e), and 6.0-6.2 (process f).

[0046] Example 4: Determination of Viral Titer

[0047] The hemagglutination titer (HA) and median lethal dose (TCID) of Newcastle disease virus prepared by the fully suspended cell culture method in Example 3 and the chicken embryo method disclosed in Chinese Patent CN107287168A were determined. 50 ) and chicken embryo median lethal dose (EID) 50 The specific method is as follows.

[0048] HA titer determination: determined according to the current Chinese Veterinary Pharmacopoeia.

[0049] TCID 50 Titer determination: The harvested virus solution was serially diluted 10-fold, and 10-fold titers were taken. -6 10 -7 10 -8 10 -9 Four dilutions were seeded into 96-well cell culture plates containing a confluent monolayer of BHK-21 cells, with 8 wells seeded for each dilution. Cells were incubated at 37°C in a 5% CO2 incubator for 3-4 days. Cytopathic effects were observed, and the number of wells with cytopathic effects for each dilution was recorded. TCID was calculated using the Reed-Muench method. 50 .

[0050] EID 50 Titer determination: Take the virus to be tested and dilute it with physiological saline for 10... -7 10 -8 10 -9 10 -10 The strains were diluted and inoculated into the allantoic cavity of 9-11 day old SPF chicken embryos, with 5 embryos inoculated at each dilution. Each embryo was inoculated with 0.1 ml of the solution. After paraffin sealing, the embryos were incubated at 37°C. Dead embryos were placed at 4°C and observed continuously for 120 hours daily. The allantoic fluid HA of all embryos was measured, and the EID of the strain was calculated using the Reed-Muench method.50 .

[0051] The results are shown in Table 2.

[0052] Table 2. Effect of culture pH during the virus amplification phase on virus titer.

[0053] The results showed that compared with the chicken embryo process, process d had a higher HA titer, while the viral load did not differ significantly between the two processes. Processes c, d, and e had significantly higher HA titers and viral loads than processes a and b, with HA titers of 10⁻¹¹ log² and viral loads of 8.89–9.57 lg TCID⁻¹. 50 / ml and 8.63-9.38lgEID 50 In process f (0.1 ml), compared to processes c, d, and e, the HA titer and viral titer decreased. This may be because the excessively low pH environment caused cell death, thus affecting viral amplification. This indicates that controlling the pH to 6.2-6.8 during the viral amplification stage can improve viral titer, with the optimal pH range being 6.4-6.6. The pH during viral amplification is inconsistent with that of cell growth. This may be because while lower pH negatively impacts cell growth and viability, it can promote viral infection and / or replication within cells and / or viral budding and release. Therefore, during process optimization, the pH cannot be simply controlled at the optimal pH for cell growth. This invention employs a two-stage pH control method to effectively improve viral culture titer.

[0054] Example 5: Preparation of NDV Inactivated Vaccine

[0055] Newcastle disease virus solutions prepared by process AF and chicken embryo method were separately introduced into inactivation tanks, and 10% formaldehyde solution was added. The mixture was stirred until fully combined, with a final formaldehyde concentration of 0.1%. Inactivation was carried out at 37°C for 16 hours. Four portions (volume ratio) of the completely inactivated solution with a content of 8.01 g EID were then collected. 50 Slowly add 0.1 ml of virus solution to 6 parts of white oil adjuvant while starting the motor and stirring at 4000 r / min for 30-40 min. Before stopping the stirring, add 1% thimerosal solution to make the final concentration 0.01%.

[0056] Example 6: Efficacy Test of Newcastle Disease Virus Inactivated Vaccine

[0057] Eighty 30-day-old SPF chickens were divided into eight groups of ten each. Groups 1-7 received an intramuscular injection of the vaccine prepared in Example 5, with an immunization dose of 20 μl. Group 8 served as a control and was not immunized. All experimental chickens were kept in isolation. 21 days after immunization, blood was collected from both the chickens and the control group to separate serum and determine the HI antibody titer. Simultaneously, each chicken received an intramuscular injection of 51 g EID. 50The virus was challenged with the HN1101 strain, which is disclosed in Chinese patent CN105985966A. Patients were observed for 14 days, and the incidence, mortality, and protection rates were recorded. The results are shown in Table 3.

[0058] Table 3. Effect of culture pH on immunogenicity

[0059] The antigens prepared using processes c, d, e, and f, when used to make vaccines for immunizing chicks, showed higher HI antibody titers than those prepared using processes a and b. Furthermore, the HI antibody titers from processes d, e, and f were higher than those from the chicken embryo process. The HI antibody titer results in this example, combined with the virus titer results from Example 4, indicate that controlling the pH during the virus amplification stage at 6.2-6.8 not only increases the virus titer in fully suspended cell cultures but also enhances immunogenicity. This may be because low pH affects cell and virus metabolism, altering the structure of immunogenic proteins and thus increasing immunogenicity. There was no significant difference in HI titers between processes d and e. However, considering the results from Example 4, the HA titer and virus content of process d were higher than those of process e. Therefore, the optimal pH control range for the virus amplification stage is process d, specifically 6.4-6.6.

[0060] Example 7: Preparation of a multivalent vaccine

[0061] Antigen preparation methods: Methods for preparing avian influenza virus SZ strain, infectious bursal disease rVP2 protein, and avian adenovirus (group I, type 4) Fiber-2 protein are disclosed in Chinese Patent CN108126191A. Methods for preparing recombinant proteins from the HMTp2102 region of infectious coryza (types A, B, and C) are disclosed in Chinese Patent CN110540579A.

[0062] Preparation of S protein from Infectious Bronchitis M41 strain: The S protein gene was amplified using M41 strain viral nucleic acid as a template. PCR amplification was performed using oligonucleotide primers synthesized based on the S protein gene sequence (primer sequences are shown in Table 4). The PCR product was recovered by XbaⅠ / HindⅢ restriction enzyme digestion and ligated into the linearized pcDNA3.1 vector using T4 DNA ligase to construct an expression vector for the S protein. The ligation product was transformed into DH5α Escherichia coli competent cells. Positive plasmids were extracted and transfected into Expi-CHO cells. Feed culture was performed on the second day after transfection, and samples were collected on day 7 for SDS-PAGE identification. The protein was expressed in the secretory supernatant. The expression product was harvested on day 9 after transfection. Purification was performed using conventional nickel affinity chromatography on Ni Sepharose 6Fast Flow medium. The elution product was collected. The Infectious Bronchitis S antigen protein was obtained by dialyzing with PBS buffer at 4°C.

[0063] Table 4 Primers for the S protein gene of infectious bronchitis strain M41 S-F 5'-CTAGTCTAGAATGTAG TGCTGCTTTGTATGACAG-3' S-R 5'-CCCAAGCTTTTAAACAGAC TTTTTAGGTC TGTTTTG-3'

[0064] Vaccine preparation method: Trivalent, quadrivalent, and pentavalent vaccines were prepared by mixing the inactivated Newcastle disease virus solution. Vaccine 1 is a Newcastle disease and bronchitis trivalent inactivated vaccine prepared in this application with equal volumes of Newcastle disease virus, infectious bronchitis S protein, and avian influenza (H9 subtype) virus solution; Vaccine 2 is a Newcastle disease virus prepared in this application with avian influenza (H9 subtype) virus solution, avian adenovirus (group I, type 4) Fiber-2 protein, and infectious coryza (types A, B, and C) HMTp210. Vaccine 2 is a tetravalent inactivated vaccine for avian influenza, bronchitis, and nasal infections prepared by mixing recombinant proteins in equal volumes. Vaccine 3 is a tetravalent inactivated vaccine for avian influenza, bronchitis, and nasal infections prepared in this application by mixing Newcastle disease virus, avian influenza (H9 subtype) virus, infectious bursal disease rVP2 protein, and avian adenovirus (group I, type 4) Fiber-2 protein in equal volumes. Vaccine 4 is a pentavalent inactivated vaccine for avian influenza, bronchitis, and nasal infections prepared in this application by mixing Newcastle disease virus, infectious bronchitis S protein, avian influenza (H9 subtype) virus, infectious bursal disease rVP2 protein, and avian adenovirus (group I, type 4) Fiber-2 protein in equal volumes. The antigens were mixed in proportion and added to the white oil adjuvant. Simultaneously, the motor was started and stirred at 4000 rpm for 30-40 minutes. Before stopping the stirring, 1% thimerosal solution was added to achieve a final concentration of 0.01%. Specific proportions are shown in Table 5.

[0065] Table 5. Composition ratio of multivalent vaccines

[0066] Example 8: Efficacy Trial of Gen VII Newcastle Disease Combined Vaccine

[0067] Fifty 30-day-old SPF chickens were divided into 5 groups of 10 each. Groups 1-4 received intramuscular injections of vaccines prepared in Example 7 (vaccines 1-4) at a dose of 20 μl each. Group 5 served as a control and was not immunized. All experimental chickens were kept in isolation. 21 days after immunization, blood was collected from both the chickens and the control group to separate serum and determine the HI antibody titer. Simultaneously, each chicken received an intramuscular injection of 51 g EID. 50 The virus was challenged with the HN1101 strain, and the incidence, mortality, and protection rates were recorded for 14 days. The results are shown in Table 6.

[0068] Table 6 Results of the combined vaccine's partial efficacy trial against Newcastle disease.

[0069] The results showed that vaccines 1-4 all produced high levels of HI antibodies against Newcastle disease virus on day 21 post-immunization, and compared with the control, the immunized group provided complete protection against virulent virus challenge. This confirms good compatibility between different antigens, and the Newcastle disease virus antigen prepared in this application can be used to prepare combination vaccines with other chicken pathogen antigens.

[0070] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

Claims

1. A method for culturing Newcastle disease virus in BHK-21 fully suspended cells, characterized in that, The method employs a two-stage pH control, wherein the pH is controlled at 7.2-7.4 during the BHK-21 cell growth stage and at 6.2-6.8 during the Newcastle disease virus amplification stage, preferably at 6.4-6.

6.

2. The method according to claim 1, characterized in that, The two-stage pH control is achieved by introducing CO2 or adding sodium bicarbonate during the culture process.

3. The method according to claim 1, characterized in that, Includes the following steps: Step (1) Establishment of Newcastle disease virus seed batch; Step (2) BHK-21 fully suspended cell culture; Step (3) NDV virus amplification and harvesting.

4. The method according to claim 3, characterized in that, The establishment of the Newcastle disease virus seed batch in step (1) includes inoculating the Newcastle disease virus into the BHK-21 fully suspended cells at an MOI of 0.01-0.1, adding trypsin at a final concentration of 20-50 mg / L, culturing in a 37°C, 5% CO2 shaking incubator for 72 h, harvesting the virus solution, and passaged 3-4 times to use as the reactor seed virus.

5. The method according to claim 3, characterized in that, Step (2) of BHK-21 full suspension cell culture includes thawing frozen cells from a liquid nitrogen tank, gradually scaling up to obtain a sufficient quantity of BHK-21 cells, and maintaining a cell density of 1.0 × 10⁻⁶ cells. 6 -1.5×10 6 Cells were inoculated at a rate of 1 / ml into the bioreactor. The bioreactor temperature was set at 37℃. The dissolved oxygen (DO) value was controlled at 30%-50% by adjusting the stirring speed and aeration rate. The pH was controlled at 7.2-7.4 by introducing CO2 or adding sodium bicarbonate. The cells were cultured for 72-96 hours, and the cell density and viability were monitored.

6. The method according to claim 3, characterized in that, Step (3) includes when the cell density is ≥1.0×10 7 Add virus amplification medium when the cell density is 5.0 × 10⁶ cells / ml to adjust the cell density to 5.0 × 10⁶ cells / ml. 6 -6.0×10 6 Inoculate with chicken Newcastle disease virus seed at a concentration of 1000 / ml and MOI = 0.01-0.1, while adding trypsin at a final concentration of 20-50 mg / L. Set the reactor temperature to 32-35℃ and dissolved oxygen to 30%-50%. Control the pH to 6.2-6.8 by introducing CO2 or adding sodium bicarbonate, preferably 6.4-6.

6. Harvest the virus solution after culturing for 96-120 hours.

7. The method according to claim 3, characterized in that, Steps (2) and (3) were carried out in a 14L bioreactor.

8. The method according to claim 3, characterized in that, The Newcastle disease virus in chickens is the N7a strain.

9. Newcastle disease virus in chickens prepared by the method of any one of claims 1-8.

10. The use of the Newcastle disease virus prepared by the method of any one of claims 1-8 in Newcastle disease inactivated vaccines, Newcastle disease and bronchitis trivalent inactivated vaccines, Newcastle disease and bronchitis adenovirus quadrivalent inactivated vaccines, Newcastle disease and bronchitis adenovirus quadrivalent inactivated vaccines, and Newcastle disease and bronchitis adenovirus pentavalent inactivated vaccines.

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